throbber
PCI‘
`
`International Bureau
`WORLD INTELLECTUAL_PROPERTY ORGANIZATION
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 4 1
`C07K 7/10, 7/34, A61K 37/02
`A61K 37/28
`
`(11) International Publication Number:
`
`W0 87/ 06941
`
`A1
`
`(43 International Publication Date:
`)
`
`19 November 1987 (19.11.87)
`
`(21) International Application Number:
`
`PCT/USS7/01005
`
`(22) International Filing Date:
`
`5 May 1987 (05.05.87)
`
`(31) Priority Application Number:
`
`859,928
`
`(81) Designated States: AT (European patent), BE (Euro-
`pean patent), CH (European patent), DE (European
`patent), FR (European patent), GB (European pa-
`tent), IT (European patent), JP, LU (European pa-
`tent), NL (European patent), SE (European patent).
`
`(32) Priority Date:
`‘
`(33) Priority Country:
`
`5 May 1986 (05.05.86) Published
`With international search report.
`
`US
`
`nue, N.W., Suite 300, Washington, DC 20036 (US).
`
`
`(71) Applicant: THE GENERAL HOSPITAL CORPORA-
`TION [US/US]; Fruit Street
`(Bar-3), Boston, MA
`02114 (US).
`
`(72) Inventor: HABENER, Joel ; 217 Plymouth Road, New-
`ton Highlands, MA 02161 (US).
`
`(74) Agents: GOLDSTEIN, Jorge, A. et al.; Saidman,
`Sterne, Kessler & Goldstein, 1225 Connecticut Ave-
`
`(54) Title: INSULINOTROPIC HORMONE
`
`(57) Abstract
`
`A fragment of glucagon-like peptide I (GLP—l) has been found to be an insulinotropic hormone. This insulinotropic
`hormone comprises amino acid residues 7-37 of GLP—1. The insulinotropie hormone is useful as a potential therapy for Di-
`abetes Mellitus.
`
`MYLAN INST. EXHIBIT 1039 PAGE 1
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`MYLAN INST. EXHIBIT 1039 PAGE 1
`
`MYLAN INST. EXHIBIT 1039 PAGE 1
`
`

`

`FOR THE PURPOSES 0F INFORMAHON ONLY
`
`Codes used to identif}r States party to the PCT on the front pages ofpamphlets publishing international appli-
`cations under the PCT.
`
`Madagascar
`
`AT Austria
`AU Australia
`BB Barbados
`BE ' Belgium
`36 Bulgaria
`3.] Benin
`BR Brazil
`CF Central African Republic
`CG Congo
`CH7 Switzerland
`CM Cameroon
`DE Germany, Federal Republic of
`DK’ Denmark
`FI
`Finland
`
`France
`Gabon
`United Kingdom
`Hungary
`Italy
`Japan
`Democratic People’s Republic
`of Korea
`Republic of Korea
`Liechtenstein
`Sri Lanka
`Luxembourg
`Monaco
`
`Mali
`Mauritania
`Malawi
`Netherlands
`Norway
`Romania
`Sudan
`Sweden
`Senegal
`Soviet Union
`Chad
`Togo
`United States of America
`
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`

`

`WO 87/06941
`
`,
`
`PCT/USS7/01005
`
`INSULINOTROPIC HORMONE
`
`‘ BACKGROUND OF THE INVENTION
`
`Field of the Invention
`
`.
`
`.This invention is directed to the discovery that
`
`certain peptide fragments of
`
`the prehormone, proglu-
`
`cagon, possess hormonal activities and can be used to
`
`stimulate the synthesis and secretion of the hormone,
`
`insulin. These peptide fragments are useful
`
`in ther-
`
`apy for the disease Diabetes mellitus.
`
`Description of the Background Art
`The endocrine secretions of the pancreatic islets
`
`are under
`
`complex control not only by blood-borne
`
`metabolites
`
`(glucose,
`
`amino acids,
`
`.catecholemines,
`
`etc.), but also by local paracrine influences.
`The
`major pancreatic islet hormones (glucagon,
`insulin and
`somatostatin)
`interact
`amongst
`their
`specific cell
`types
`(A, B; and D cells,
`respectively) to modulate
`secretory responses mediated by the metabolites. Al-
`
`though insulin secretion is predominantly controlled
`by blood levels of glucose, glucagon and somatostatin
`stimulate and inhibit glucose—mediated insulin secre-
`
`tory responses, respectively.
`
`In addition to the pro-
`
`MYLAN INST. EXHIBIT 1039 PAGE 3
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`
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`

`

`WO 87/06941
`
`_
`
`PCT/USS7/01005
`
`insulin se-
`posed interislet paracrine regulation of
`cretion,
`there is evidence to support the existence of
`
`insulinotropic factors in the intestine. This concept
`originates from the~observations that glucose taken
`orally is a much more potent stimulant of insulin se-
`cretion than is a comparable amount of glucose given ,
`
`intravenously.
`
`The human hormone, glucagon,
`
`is a 29-amino acid
`
`the pan—
`peptide hormone produced in the A-cells of
`creas.
`The hormone belongs to a multi-gene family of
`
`structurally related peptides that
`
`include secretin,
`
`gastric inhibitory peptide, vasoactive intestinal pep-
`tide and glicentin.
`These peptides variously regu-
`late carbohydrate metabolism, gastrointestinal mobil—
`ity and secretory processing. The principal recognized
`actions of pancreatic glucagon, however, are to pro-
`mote glycogenolysis and gluconeogenesis, resulting in
`an elevation of blood sugar levels.
`In this regard,
`
`the actions of glucagon are counterregulatory to those
`
`of
`
`insulin and :may contribute to the hyperglycemia
`
`(Lund, P. K. e;
`
`that accompanies Diabetes mellitus
`
`al., Proc. Natl. Acad. Sci., USA, 12: 345-349 (1982)).
`Glucagon has been found to be capable of binding
`to specific receptors which lie on the surface of in-
`sulin producing cells. Glucagon, when bound to these
`receptors, stimulates the rapid synthesis of CAMP, by
`these cells. CAMP,
`in turn, has been found to stimul-
`ate insulin expression (Korman, L.Y. §§_§l., Diabetes,
`gg:717-722 (1985)).
`Insulin acts to inhibit glucagon
`synthesis (Review of Medical Physiology, Ganong, W.F.,
`1979 Lange Publications, Los Altos, California (p.
`
`273).
`
`Thus the expression of glucagon is carefully
`
`MYLAN INST. EXHIBIT 1039 PAGE 4
`
`MYLAN INST. EXHIBIT 1039 PAGE 4
`
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`

`

`WO 87/06941
`
`,
`
`PCT[U587[01005
`
`regulated by insulin, and ultimately by the serum glu-
`cose level.
`
`The glucagon gene is initially tranlated from a
`
`630 base pair precursor to form the polypeptide, pre-
`
`proglucagon (Lund gt a1; (1982)). This polypeptide is
`
`subsequently processed to form proglucagon. Patzelt,
`
`C. et al;, Nature, ggg: 260-266 (1979), demonstrated
`
`that proglucagon was subsequently cleaved into gluca-
`
`Subsequent work by
`second. polypeptide.
`gen and a
`Lund, P. K. gt al;, Lopez L. C. gt alL, and Bell, G.
`
`I. et a1.,
`
`(Nature) 302:716-718 (1983) demonstrated
`
`that the proglucagon molecule was cleaved immediately
`
`Studies of
`after lysine-arginine dipeptide residues.
`proglucagon produced by
`channel catfish (Ictalurus
`punctata) indicated that'glucagon from this animal was
`
`also ,proteolytically cleaved. after adjacent
`
`lysine-
`
`arginine and arginine-arginine dipeptide residues (An-
`
`drews, P. C. gt gl;, J. Biol. Chem., ggg: 3910-3914
`
`(1985)). Lopez, L. C. 25 Ei-r (Proc. Natl. Acad. Sci.
`
`gs; 89:5485-5489 (1983)), and Bell, G. I. et al, dis-
`
`covered the mammalian proglucagon was cleaved at 1y-
`
`sine-arginine or arginine arginine dipeptides,
`
`and
`
`demonstrated that
`
`the proglucagon molecule contained
`
`three discreet and highly homologous peptide molecules
`
`which were designated glucagon, glucagon-like protein
`
`1 (GLP—1) and glucagon-like protein 2
`
`(GLP-Z).> Lopez
`
`gt 5;; concluded that glucagon-like protein 1 was 37
`
`amino acid residues long and that glucagon-like pep-
`tide 2 was 34 amino acid residues long.
`Analogous
`
`studies on the structure of rat preproglucagon reveal—
`
`ed a similar pattern of proteolytic cleavage between
`
`adjacent
`
`lysine-arginine or arginine-arginine dipep‘
`
`Wl
`
`‘
`
`MYLAN INST. EXHIBIT 1039 PAGES
`
`MYLAN INST. EXHIBIT 1039 PAGE 5
`
`MYLAN INST. EXHIBIT 1039 PAGE 5
`
`

`

`WO 87/06941
`
`.
`
`_
`
`PCT/USS7/0 1005
`
`tide residues, resulting in the formation of glucagon,
`GLP-1 and GLP-2
`(Heinrich,
`(L gt alL, Endocrinol.,
`11;: 2176-2181 (1984)).
`Human rat, bovine, and ham-
`ster sequences of GLP-1 have been found to be identi—
`cal
`(Ghiglione, M. et al., Diabetologia, 21:599-600
`
`(1984)).
`regarding
`The conclusion reached by Lopez st 31.
`the size of GLP-1 was confirmed by the work of Utten-
`thal, L.O. 35 al; (J. Clin. Endocrinol. Metabol., fil:
`472-479 (1985)). Uttenthal £E.él; examined the molec-
`ular forms of GLP-1 which were present
`in the human
`pancreas. Their research shows that GLP-1 and GLP-2
`are'present in the pancreas as 37 amino acid and 34
`amino acid peptides, respectively.
`The similarity between GLP-1 and glucagon suggest-
`ed to early investigators that GLP-l might have bio-
`logical activity. Although some investigators found
`that GLP-l could induce rat brain cells to synthesize
`cAMP
`(Hoosein, N.M. et al., Febs Lett.
`178:83-86
`(1984)), other
`investigators failed to identify any
`physiological role for GLP;1 (Lopez, L. C. 23 21;).
`The failure to identify any physiological
`role for
`GLP-l caused some
`investigators to question whether
`GLP-l was in fact a hormone and whether the related-
`ness between glucagon and GLP-1 might be artifactual
`
`(Ghiglione, M. 23 51;).
`an
`reveals
`the prior art
`Thus,
`in conclusion,
`awareness of the processing of a glucagon hormone pre-
`cursor into a set of peptides sharing extensive homo-
`logy.
`It has been widely assumed by those of skill in
`the art that these highly related glucagon-like pep-
`tides would have a biological activity. Nevertheless,
`extensive investigations designed to elucidate the
`
`MYLAN INST. EXHIBIT 1039 PAGE 6
`
`MYLAN INST. EXHIBIT 1039 PAGE 6
`
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`
`

`

`WO 87/06941
`
`,
`
`PCT[USS7/01005
`
`biological effects of these molecules had been unsuc-
`cessful.
`
`
`SUMMARY_ OF THE INVENTION
`
`The hormone glucagon is known to be synthesized as
`
`a high molecular weight precursor molecule which is
`
`subsequently proteolytically cleaved into three pep—
`
`tides: glucagon, glucagon—like peptide 1
`
`(GLP-1) and
`
`GLP-l has 37 amino
`(GLP-Z).
`glucagon-like peptide 2
`This
`invention dis-
`acids in its unprocessed form.
`closes that
`the unprocessed GLP-1 is naturally con-
`
`verted to a 31 amino acid long peptide (7-37 peptide)
`
`This processing
`having amino acids 7-37 of GLP-1.
`occurs in the pancreas and the intestine.
`The 7-37
`
`peptide is an insulinotropic hormone which had not
`
`The hormone’s activity
`previously been described.
`appears to be specific for the pancreatic beta cells
`
`where it appears to induce the biosynthesis of insu-
`
`lin.
`
`The unprocessed GLP-l peptide is essentially
`
`unable to mediate the induction of insulin biosynthe-
`
`sis.
`
`The
`
`insulinotropic hormone
`
`is useful
`
`in the
`
`study of the pathogenesis of maturity onset diabetes
`
`mellitus, a condition in which the dynamics of insulin
`secretion are abnormal. Moreover,
`the insulinotropic
`
`hormone is useful in therapy for this disease.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`Figure 1 shows the DNA structure and corresponding
`
`amino acid sequence of human, rat and hamster prepro-
`
`glucagons.
`
`The preproglucagon precursor
`
`is proteo-
`
`lytically cleaved at sites indicated by circles.
`
`—- ,,
`
`MYLAN INST. EXHIBIT 1039 PAGE 7
`
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`
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`

`

`wo 87106941
`
`7
`
`PCT/U587/01005
`
`Figure 2
`
`shows
`
`the effect of GLP-1 peptides on
`
`insulin mRNA levels in rat insulinoma cells.
`
`the effects of GLP-1 peptides on
`shows
`Figure 3
`' angiotensingen mRNA levels in rat insulinoma cells.
`Figure 4
`shows
`the effects of GLP-1 peptides on
`
`actin mRNA levels in rat insulinoma cells.
`
`Figure 5 shows the effect of GLP-1 (1-37) on pro-
`
`lactin mRNA levels in GH4 cells.
`
`Figure 6 shows the effects of GLP-1 (1-37) on ACTH
`
`mRNA levels in AtT-ZO cells.
`
`DESCRIPTION OF THE PREFERRED EMBODIMENTS
`
`.Peptide moieties (fragments) chosen from the de-
`termined amino acid sequence of human GLP-l conStitute
`
`in the development comprising the
`the starting point
`The amino acid sequence for GLP—l
`present invention.
`has been reported by several researchers (Lopez, L. C.
`
`(Nature) 323:716-
`(1983); Bell, G. I. gt 3;;,
`22 31;
`718 (1983); Heinrich, G. 33 31; (1984); Ghiglione, M.
`
`The structure of the preproglucagon
`(1984)).
`g; EL;
`gene and its corresponding amino acid sequence
`is
`
`shown in Figure 1.
`
`This figure further displays the
`
`proteolytic processing of the precursor gene product
`
`into glucagon and the two glucagon-like peptides.
`
`As
`
`used herein,
`
`the notation GLP-l
`
`(l-37)
`
`refers to a
`
`GLP-l polypeptide having all
`
`amino
`
`acids
`
`from 1
`
`Similarly,
`(C-terminus).
`-through 37
`(N—terminus)
`GLP-l
`(7-37) refers to a GLP-l polypeptide having all
`amino acids from 7
`(N-terminus)
`through 37
`(Ceterm-
`
`inus).
`
`In one embodiment,
`
`the peptide fragments are syn-
`
`thesized by the well-known solid phase peptide syn-
`
`MYLAN INST. EXHIBIT 1039 PAGE 8 ~
`
`MYLAN INST. EXHIBIT 1039 PAGE 8
`
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`

`

`WO 87/06941
`
`'
`
`PCTIUSS7/01005
`
`_ thesis described by Merrifield, J: M., Chem. Soc., §§:
`
`2149 (1962), and Stewart and Young, Solid Phase Pep-
`
`(Freeman, San Francisco, 1969), pages
`tide Synthesis
`27-66, which are incorporated by reference herein.
`However,
`it is also possible to obtain fragments of
`
`the proglucagon polypeptide or of GLP-1 by fragmenting
`
`the naturally-occurring amino acid sequence, using,
`
`for example,
`
`a proteolytic enzyme.
`
`Further,
`
`it is
`
`possible to obtain the desired fragments of the pro—
`
`glucagon peptide or of GLP-1 through the use of recom-
`
`binant DNA technology, as disclosed by Maniatis, T. g;
`
`A Laboratory Manua , Cold
`al., Molecular Biology:
`Spring Harbor, NY 1982, which is hereby incorporated
`
`by.reference.
`
`The invention pertains to a peptide fragment which
`
`is insulinotropic and is derivable from a naturally-
`occurring amino acid sequence.
`
`The invention comprises a peptide fragment having
`
`the following amino acid sequence:
`
`His—Ala-Glu-Gly-Thr-Phe-Thr—Ser-Asp-
`Val-Ser-Ser-Tyr-Leu—Glu-Gly-Gln-Ala-
`Ala-Lys-Glu-Phe-Ile-AlafTrp-Leu-Val-
`Lys-Gly-Arg-Gly
`
`and functional derivatives thereof,
`
`these fragments
`
`and functional derivatives being substantially free of
`
`natural contaminants and having insulinotropic activ-
`
`ity.
`
`of particular interest are peptides of the follow-
`
`ing formula:
`
`MYLAN INST. EXHIBIT 1039 PAGE 9
`
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`

`WO 87/06941
`
`PCT[U587[01005
`
`(1) H2N--X--C0--Rl
`
`wherein R
`
`1.
`
`is OH, OM or -NR2R3;
`
`M is a pharmaceutically acceptable
`cation or a lower
`(Clgc ) branched or unbranched
`6
`alkyl group; R2 and R
`are the same or different
`and selected from the group consisting of hydrogen
`
`and a lower
`group; and
`
`(Cl-C6) branched or unbranched alkyl
`
`X is the amino acid sequence or pep-
`
`tide fragment described above;
`
`(2)
`
`The acid addition salts thereof; and
`
`The protected or partially protected deriva-
`(3)
`tives thereof.
`'
`
`The invention further pertains to a method for
`
`enhancing the expression of insulin which comprises:
`providing to a mammalian pancreatic B-type
`islet cell an effective amount of the insulinotropic
`
`peptides disclosed above.
`
`Included within the scope of the present invention‘
`
`are those amino acid sequences in the above peptides
`
`insulinotropic
`functioning as
`which are capable of
`hormones.
`Included as well are the use of additional
`
`amino acid residues added to enhance coupling to car—
`
`rier protein or amino acid residues added to enhance-
`
`the insulinotropic effect.
`
`A material is said to be
`
`"substantially free of natural contaminants" if it has
`
`been substantially purified from materials with which
`
`it is normally and naturally found.
`
`Examples of nat-
`
`ural contaminants with which GLP-l
`
`(7-37) might be
`
`MYLAN INST. EXHIBIT 1039 PAGEJJD,
`
`MYLAN INST. EXHIBIT 1039 PAGE 10
`
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`

`

`WO 87/06941
`
`_
`
`PCT/USS7/01005
`
`other peptides,'carbohydrates, gly-
`associated are:
`cosylated peptides, lipids, membrane, etc.
`A material
`is also said to be substantially free of natural con—
`taminants if these contaminants are substantially ab-
`sent from a sample of the material.
`The interchangeable terms "peptide fragment" and
`"peptide moiety" are meant
`to include both synthetic
`and naturally-occurring amino acid sequences derivable
`from a naturally occurring amino acid sequence.
`A peptide is said to be "derivable from a natural-
`ly-occurring amino acid sequence“ if it can be obtain-
`ed by fragmenting a naturally-occurring sequence, or
`if'it can be synthesized based upon a knowledge of the
`sequence of the naturally occuring amino acid sequence
`or of the genetic material
`(DNA or RNA) which encodes
`
`this sequence.
`to polypeptides
`The
`invention further -pertains
`that,
`in addition to the chosen sequence, may contain
`or lack one or more amino acids that may not be pre-
`sent in the naturally—occurring sequence wherein such
`polypeptides are functionally similar
`to the chosen
`polypeptide.
`Such polypeptides for the present inven-
`tion, are termed "functional derivatives," provided
`that they demonstrate insulinotropic activity which is
`substantially similar to that of GLP-1 (7-37).
`An "insulinotropic activityI relates to the abil-
`ity of a substance to stimulate, or cause the stimula-
`tiOn‘of,
`the synthesis or expression of
`the hormone
`
`insulin.
`the amino acid residues
`As
`is known in the art,
`may be in their protected or unprotected form, using
`appropriate amino or carboxyl protecting groups. Use-
`
`MYLAN INST. EXHIBIT 1039 PAGE 11
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`

`wo 87/06941
`
`_
`
`_
`
`PCT/USS7/01005
`
`-10-
`
`ful cations are alkali or alkaline earth metallic ca-
`-tions (i.e., Na, K, Li,
`l/2Ca,
`l/2Ba, etc.) or amine
`
`cations
`
`(i.e.,
`
`tetraalkylammonium,
`
`trialkylammonium,
`
`where alkyl can be Cr-Clz).
`The variable length peptides may be in the form of
`
`the free amines (on the N-terminus), or acid—addition
`
`salts thereof.
`
`Common acid addition salts are hydro-
`
`halic acid salts, i.e., HBr, HI, or more preferably,
`
`HCl.
`
`The insulinotropic property of a compound may be
`determined by providing that compound to animal cells,
`or injecting that compound into animals and monitoring
`the release of immunoreactive insulin (IRI)
`into the
`media or circulatory system of the animal, respective-
`
`ly.‘ The-presence of IRI is detected through the use
`of a radioimmunoassay which can specifically detect
`
`insulin.
`
`Although .any radioimmunoassay' capable of
`
`detecting the presence of IRI may be employed, it is
`preferable to use a modification of the assay method
`of Albano,
`J.D.M., et
`a1.
`(Acta Endocrinol.
`1Q:
`
`487—509 (1972)).
`
`In this modification a phosphate/al-
`
`bumin buffer with a pH of 7.4 was employed.
`
`The in-
`
`cubation was prepared with the consecutive condition
`of 500 ul oprhosphate buffer, 56 ul of perfusate sam-
`ple or rat insulin standard in perfusate, 100 ul of
`anti-insulin
`antiserum
`(Wellcome
`Laboratories;
`1:40.000 dilution), and 100 ul of [12511 insulin, giv-
`ing a total volume of 750 ul
`in a 10 X 75-mm dispos-
`abiqulass tube. After
`incubation for 2-3 days at
`4°C,
`free insulin was separated from antibody-bound
`insulin by charcoal separation. The assay sensitivity
`
`was 1-2 uU/ml.
`
`In order to measure the release of IRI
`
`MYLAN INST. EXHIBIT 1039 PAGE 12
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`MYLAN INST. EXHIBIT 1039 PAGE 12
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`

`WO 87/06941
`
`V
`
`‘ PCT/U587/01005
`
`-11..
`
`into the cell culture medium of cells grown in tissue
`
`culture, one preferably incorporates radioactive label
`
`into proinsulin. Although any radioactive label cap—
`able of
`labelling a'polypeptide can be used,
`it is
`3
`
`preferable to use
`
`H leucine in order to obtain label-
`
`led proinsulin. Labelling can be done for any period
`
`of time sufficient to permit
`
`the formation of a de-
`
`tectably labelled pool of proinsulin molecules; how-
`
`ever, it is preferable to incubate cells in the pre-
`
`sence of radioactive label for a 60 minute time per-
`
`iod.
`
`Although any cell
`
`line capable of expressing
`
`insulin can be used for determining whether a compound
`
`has'an insulinotropic effect, it is preferable to use
`rat insulinoma cells, and especially RIN - 38 rat in-
`
`sulinoma cells.
`
`Such cells can be grown in any suit-
`
`able medium; however, it is preferable to use DME med-
`
`ium containing 0.1% BSA and 25 mM glucose.
`
`The insulinotropic property of a compound may also
`
`be determined by pancreatic infusion.
`
`The
`
`in situ
`
`(gia-
`
`isolated perfused rat pancreas preparation was a modi-
`
`fication of the method of Penhos, J. C. et al.,
`
`betes,
`
`lgz733-738 (1969)).
`
`Fasted male Charles River
`
`strain albino rats, weighing 350e600 g, were anesthe-
`
`tized with an intraperitoneal injection of Amytal Sod-
`
`ium (Eli Lilly and Co.; 160 ng/kg). Renal, adrenal,
`
`gastric, and lower colonic blood vessels are ligated.
`
`The.entire intestine was
`
`resected except
`
`for about
`
`four.cm of duodenum and the descending colon and rec-
`
`tum.- Therefore, only a small part of
`
`the intestine
`
`was perfused,
`
`thus minimizing possible interference by
`
`enteric substances with glucagon-like immunoreactiv—
`
`ity.
`
`The perfusate was a modified Krebs-Ringer bicar-
`
`__l
`
`.
`
`MYLAN INST. EXHIBIT 1039 PAGE 13
`
`MYLAN INST. EXHIBIT 1039 PAGE 13
`
`MYLAN INST. EXHIBIT 1039 PAGE 13
`
`

`

`WO 87/0694!
`
`7
`
`PCT[U587/01005
`
`-12-
`
`bonate buffer with 4% dextran T70,and 0.2% bovine ser-
`um albumin (fraction V), and was bubbled with 95% O2
`and 5% C02.
`A nonpulsatile flow, 4-channel roller
`bearing pump (Buchler polystatic, Buchler Instruments
`
`Division, Nuclear-Chicago Corp.) was used,
`
`and
`
`a
`
`switch from one perfusate source to another was accom—
`plished by switching a 3-way stopcock.
`The manner
`in
`which perfusion was performed, monitored, and analyzed
`
`followed the method of Weir, G. C. et al.
`(J. Clin.
`Investigat.
`ii:
`1403-1412
`(1974)), which is hereby
`
`incorporated by reference.
`The compounds of the present invention can be for-
`mulated according to known methods to prepare pharma-
`ceutically useful compositions, whereby GLP-l
`(7-37)
`or its functional derivatives are combined in admix-
`
`ture with a pharmaceutically acceptable carrier vehi-
`
`cle. Suitable vehicles and their formulation,
`
`inclu-
`
`sive of other human proteins, e.g. human serum albu-
`
`min, are described for example in Remington's Pharma-
`
`ceutical Sciences (16th Ed. A. Oslo Ed. Mack, Easton
`
`PA (1980)).
`
`In order to form a pharmaceutically ac--
`
`ceptable composition suitable for effective adminis-
`tration,
`such compositions will contain an effective
`
`amount of the GLP-l (7-37), or its functional derivah
`
`tives,
`hicle.
`
`together with a suitable amount of carrier ve-
`'
`
`(7-37) or its func—
`Compositions containing GLP-l
`tional derivatives may be administered intravenously,
`
`intramuscularly, or subcutaneously at dosages in the
`range of
`from about
`1 pg/kg to 1,000 ug/kg body
`weight, although a lower or higher dosage may be ad-
`ministered.
`The required dosage will depend upon the
`
`MYLAN INST. EXHIBIT 1039 PAGE 14
`
`MYLAN INST. EXHIBIT 1039 PAGE 14
`
`MYLAN INST. EXHIBIT 1039 PAGE 14
`
`

`

`WO 87/06941
`
`.
`
`.
`
`PCT[USN/01005
`
`-13—
`
`severity of the condition of the patient and upon such
`
`criteria as the patient's height, weight,
`
`sex, age,
`
`and medical history.
`For the purpose of parenteral administration,
`
`com—
`
`positions containing GLP-l
`
`(7-37) are dissolved in
`
`distilled water and the pH—value is adjusted to about
`
`6
`
`to 8.
`
`In order
`
`to facilitate the lyophilization
`
`process resulting in a suitable product, lactose could
`
`The solution is then filter
`be added to the solution.
`sterilized,
`introduced into vials, and lyophilized.
`
`The concentration of GLP-1 (7-37)
`tions may vary from lO-lZM to lO—SM.
`
`in these composi-
`
`Additional pharmaceutical methods may be employed
`to control the duration of action. Controlled release
`
`. preparations may be achieved by the use of polymers to
`
`complex or adsorb GLP-l
`
`(7-37) or its functional der-
`
`ivatives.
`
`The controlled delivery may be exercised by
`
`selecting appropriate macromolecules
`
`(for
`
`example,
`
`polyesters, polyamino acids, polyvinyl pyrrolidone,
`
`ethylenevinylacetate, methylcellulose, carboxymethyl-
`
`cellulose, and protamine sulfate) and the concentra-
`
`tion of macromolecules as well as the methods of
`
`in-
`
`corporation in order to control release. Another pos-
`
`sible method to control the duration of action by con-
`
`trolled release preparations is to incoporate GLP-l
`
`(7437)
`
`into particles of a polymeric material such as
`
`polyesters, polyamino acids, hydrogels, poly (lactic
`
`acid)_or ethylene vinylacetate copolymers. Alterna-
`tively,
`instead of
`incorporating GLP‘l
`(7-37)
`into
`
`these polymeric particles,
`
`it is possible to entrap
`
`GLP-l
`
`(7-37)
`
`in microcapsules prepared,
`
`for example,
`
`MYLAN INST. EXHIBIT 1039 PAGE 15
`
`MYLAN INST. EXHIBIT 1039 PAGE 15
`
`MYLAN INST. EXHIBIT 1039 PAGE 15
`
`

`

`WO 87/06941
`
`,
`
`PCT/USS7/01005
`
`-14-
`
`by coacervation techniques or by interfacial polymer-
`
`ization, for example, hydroxymethylcellulose or gela-
`
`tin-microcapsules and poly (methylmethacrylate) micro-
`capsules, respectively, or in colloidal drug delivery
`
`systems, for example,
`
`liposomes, albumin microspheres,
`
`microemulsions, nanoparticles, and nanocapsules or
`
`in
`
`macroemulsions.
`
`Such
`
`teachings are disclosed in
`
`Remington's Pharmaceutical Sciences (1980).
`
`SPECIFIC EXAHPLES
`
`EXAMPLE 1
`
`;Rat insulinoma cells of cell line BIN-38 were de-
`rived from a continuous islet cell line, RIN—r, which
`
`was established from a transplantable rat islet cell
`
`(Gazdar, A. F. et al., Proc. Nat'l Acad. Sci.
`
`tumor
`
`The cells were main-
`' U.S.A. 11: 3519-3523 (1980)).
`tained in DMEM (Gibco) at a glucose concentration of
`
`4,500 mg/L, and supplemented with 10% heat-inactivated
`
`fetal bovine serum (Gibco), 100 U/ml of penicillin and
`
`Incubations were carried
`100 ug/ml of streptomycin.‘
`out at 37°C in 95% air:
`5% coz. Cells grown in the
`above manner were washed and resuspended in DMEM (Gib-
`
`co) containing 0.1% bovine serum albumin and 25 mM
`
`glucose. Cells were incubated with varying concentra-
`
`tions of GLP-1
`
`(l-37), GLP-l
`
`(7-37) or GLP-l
`
`(1-36
`
`des-gly-arg amide) for six hours,
`
`following which the
`
`effects of
`
`these agents on insulin mRNA expression
`
`were determined. Cellular RNA was analyzed for insu-
`
`lin specific mRNA as follows:
`
`cellular RNA was ex-
`
`MYLAN INST. EXHIBIT 1039 PAGE 16
`
`MYLAN INST. EXHIBIT 1039 PAGE 16
`
`MYLAN INST. EXHIBIT 1039 PAGE 16
`
`

`

`WO 87/06941
`
`7
`
`PCT/USS7/01005
`
`-15-
`
`tracted from solid tumors and cells by homogenization
`in guanadine thiocyanate and sedimentation through a
`cesium chloride cushion.
`Poly A+ RNA was isolated by
`
`oligo dT cellulose chromatography (Aviv, H. et al.,
`
`Proc. Nat'l Acad. Sci. U.S.A. g3: 1408-1412 (1972)).
`
`20 ug of total RNA from each sample was fractionated
`
`by size on a 1.4% agarose gel after denaturation in
`
`glyoxal,
`
`followed by electrotransfer to a nylon mem—
`
`brane (Nytran; Schleicher and Schuell). Blotted memr
`branes were baked for two hours at 80°C under vacuum}
`prehybridized in 1M NaCl / 1% SDS/ 10% Dextran sulfate
`at 50°C overnight and hybridized at the same tempera-
`
`the labelled probes
`ture for 24 h after addition of
`(3-5 x 105 cpm/ml);
`they were then washed, at 55°C
`twice in 1 x ssc (0.15 M'Nac1 / 0.01514 Na citrate)‘/
`1% SDS), and exposed to X—ray film for varying times
`at
`-70°C with an intensifying screen.
`In all cases
`the concentration of peptides was 10‘7M.
`
`The result of this experiment
`
`is shown in Figure
`
`2. Lanes l-3 (control cells), 4-6 (GLP-l (l-37)), 7-9
`
`GLP-l
`
`(7-37), 10-12 (GLP-lcl-BG des- gly arg -amide)
`
`shows the amount of
`insulin specific mRNA produced.
`Triplicate experimental results are presented for each
`
`peptide.
`
`Using a microdensitometer the relative amounts of
`
`insulin specific mRNA were determined.
`
`This experi-
`
`ment revealed that, at equal peptide concentrations,
`
`stimulated insulin gene expression to
`(7-37)
`SLR-l
`more than 3 times the level found in control
`(untreat—
`
`ed) cells.
`
`,1
`
`MYLAN INST. EXHIBIT 1039 PAGE 17
`
`MYLAN INST. EXHIBIT 1039 PAGE 17
`
`MYLAN INST. EXHIBIT 1039 PAGE 17
`
`

`

`W0 87/0694}.
`
`PCTIUSS7/01005
`
`-15-
`
`EXAMPLE 2:
`
`Rat
`insulinoma cells of cell
`line RIM-38 were
`grown in DME medium as described in Example 1. After
`incubation with 10‘7M GLP-l
`(1—37, GLP-l
`(7-37) and
`GLP-1
`(1-36),
`the concentrations of
`insulin in the
`cell culture mediums were determined by radioimmun-
`assay (as described above).
`Insulin protein levels
`were determined after incubation for six hours.
`The
`
`results of this experiment are shown in Table 1.
`
`TABLE 1
`
`PEPTIDE ADDED
`
`Insulin Produced
`(uUnits/ML)
`
`None
`
`.
`
`GIP-l (1-37)
`
`EXAMPLE 3:
`
`.
`
`2800
`
`5000
`
`The pancreas or live rat was perfused with vary-_
`ing concentrations of GLP-1 (1-37) and GLP-1 (7-37) as
`described above. At one minute intervals, rat serum
`
`insulin levels
`in picograms/ml were determined by
`radioimmunassay (as described above).
`The results of
`this experiment are shown in Table 2. Perfusions were
`7M,
`5 x
`done using peptide concentrations of
`5 x 10-
`‘12 M.
`5 x 10‘11M and 5 x 10
`lOfBM, and 5 x lO-loM,
`Peptides were added after the zero minute serum value
`had been determined.
`
`MYLAN INST. EXHIBIT 1039 PAGE 18
`
`MYLAN INST. EXHIBIT 1039 PAGE 18
`
`MYLAN INST. EXHIBIT 1039 PAGE 18
`
`

`

`WO 87/06941
`
`,
`
`PCT/USS7/01005
`
`-17-
`
`GLP-l
`
`(1-37) was found to mediate a 3.4-fold in-
`
`crease in serum insulin concentrations when perfused
`7M; at
`into rat pancreas at a concentration of 5 x 10-
`a concentration of 5‘x 10-8
`M this peptide was capable
`of mediating only a 2—fold increase in serum insulin
`levels. At a concentration of 5 x lO_10M this peptide
`was found to mediate only a 20% increase in serum in-
`
`sulin levels.
`
`GLPfl
`
`(7-37) was found to be capable of stimulat-
`
`ing a l32-fold increase in insulin levels when pro-
`vided to rat pancreas at a concentration of 5 x 10‘7M.
`At a lO—fold lower concentration (5 x lO-8M) this pep-
`tide was capable of directing a 21-fold increase in
`the serum. concentration of
`insulin.
`At a- concen-
`tration of 5 xth-loM, GLP-l
`(7-37) was
`found to be
`capable of mediating' an increase‘
`in serum insulin
`levels
`(32-fold).
`Even at a concentration of
`5 x
`lo-llM, GLP-l
`(7-37) delivered a 15-fold increase in
`insulin levels whereas GLP-l
`(1-37) was without
`
`effect.
`
`This experiment
`shows
`that GLP-l
`(7-37)
`is more
`than 1,000-fold more potent than GLP-l (1-37) in stim-
`
`ulating insulin expression in vivo.
`
`In addition,
`
`the
`
`GLP-l peptides had no effects on the release of the
`
`peptide hormones glucagon and somatostatin in these
`same experiments.
`Thus,
`the stimulatory effects of
`
`GLP-1 are specific for the beta cells and do not act
`
`on pancreatic alpha or delta cells.
`
`MYLAN INST. EXHIBIT 1039 PAGE 19
`
`MYLAN INST. EXHIBIT 1039 PAGE 19
`
`MYLAN INST. EXHIBIT 1039 PAGE 19
`
`

`

`WO 87/06941
`
`.
`
`PCT/USS7/01005
`
`-13..
`
`Table 2
`
`Insulin Produced (picograms/ml) at
`'
`Peptide Concentration
`
`Time
`(Minutes)
`
`5x10‘7M 5x10‘3M, 5x10‘10M 5x10‘11M 5x10‘12M
`
`GLP-l
`(7-37)
`
`012-1
`(1-37)
`
`.
`
`o
`1
`2
`3
`
`0
`1
`2
`3
`
`50
`6600
`4700
`1700
`
`1400
`4700
`2900
`2200
`
`925
`20,700
`10,500.
`4,000
`
`3,000
`6,000
`2,000
`2,000
`
`205
`7400
`1300
`750
`
`500
`600
`640
`430
`
`.
`
`160
`2400
`1700
`1900
`
`340
`100
`230
`340
`
`50
`50
`so
`98
`
`50
`50
`160
`so
`
`;.
`'
`
`MYLAN INST. EXHIBIT 1039 PAGE 20
`
`MYLAN INST. EXHIBIT 1039 PAGE 20
`
`MYLAN INST. EXHIBIT 1039 PAGE 20
`
`

`

`WO 87/06941
`
`_
`
`PCT[U587/01005
`
`-19-
`
`EXAMPLE 4:
`
`In order
`
`to determine whether glucagon-like pro-
`
`levels
`teins were capable of affecting cellular CAMP
`the effects of GLP¥1~57—37) and GLP—1
`(1-37) on CAMP
`levels iJi RINS-38 insulinoma cells was determined.
`
`Cells were grown as described in Example 1,
`
`in 26 well
`
`culture dishes. Varying amounts of glucogon—like pep-
`
`tides were added to culture wells in triplicate. Af-
`
`ter permitting incubation for§ 10 minutes
`
`the total
`
`cell media was examined for CAMP, and the concentra—
`
`tion of cAMP was determined.
`
`The results of this ex-
`
`periment are shown in Table 3.
`
`20 ul from'each cul-
`
`ture well was assayed.
`
`Peptide.
`
`Concentration
`
`Table 3
`_
`pMOLES or CAMP pRouucao‘
`
`Expt
`
`Expt
`
`
`(M)
`I
`II
`
`o
`
`10'6
`
`10‘7
`
`10‘8
`10‘9
`
`10'10
`
`-19711
`
`-
`
`140
`
`400
`
`370
`
`494
`515
`
`253
`
`533
`
`91
`
`170
`
`120
`
`160
`100‘
`
`90
`
`90
`
`.
`
`.
`
`I
`
`This experiment reveals that GLP-l (7-37) was cape
`
`able of stimulating cAMP levels even when present at a
`
`MYLAN INST. EXHIBIT 1039 PAGE 21
`
`MYLAN INST. EXHIBIT 1039 PAGE 21
`
`MYLAN INST. EXHIBIT 1039 PAGE 21
`
`

`

`WO 87106941
`
`_
`
`PCT/USS7/01005
`
`L20—
`
`concentration of lo‘llM.
`
`The increase in cAMP levels
`
`is an indication that GLP-l
`
`(7-37)
`
`is capable of
`
`in-7
`
`teracting with cellular receptors.
`
`EXAMPLE 5
`
`In order to demonstrate that the effects of GLP-1
`
`(1-37), GLP-l
`
`(1-36) and GLP-1
`
`(7-37) were specific
`
`for insulin, and were not capable of inducing-or pro-
`
`voking .non-specific gene expression,
`
`the effect of
`
`these peptides on the levels of actin and angiotens-
`
`inogen mRNAs were conducted.
`
`RIN-38 insulinoma cells
`
`were grown as described in Example 1 and incubated in
`
`(7-37), or GLP-l
`the presence of GLP-1 (l-37), GLP-l
`(l—36) des-Gly arg (Peninsula Laboratories).
`In all
`cases the concentration of peptides was 10’7M.
`Incu-
`bations were for six hours. Messenger RNAs specific
`
`for insulin, actin, or angiotensinogen were identified
`
`by Northern hybridization as described in Example 1.
`
`The results of this experiment are shown in Figure 2
`(insulin mRNA); Figure 3
`(anginotensinogen mRNA)i and
`
`Figure 4 (actin mRNA).
`mRNA levels were determined in_
`arbitrary densitometric units obtained from scanning

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